Techniques for acquiring information about biomolecules, such as nucleic acids and proteins, are applied in many different disciplines, including various branches of medical science. Many of these techniques include the use of fluorescent labels to generate detectable signals. For example, one class of fluorescent dyes that has been developed includes energy transfer fluorescent dyes. In general, energy transfer processes involving these dyes include dipole-dipole resonance interactions between donor and acceptor moieties that are associated with the same or different biomolecules. In these processes, when donor and acceptor moieties are positioned within sufficient proximity and with proper orientations relative to one another, energy emitted from donor moieties is absorbed by acceptor moieties. Detectable signals are produced when this absorbed energy causes the acceptor moieties to fluoresce.
Exemplary approaches to nucleic acid analysis that commonly utilize energy transfer fluorescent dyes include hybridization-based assays, such as nucleic acid amplification procedures (e.g., Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA), and Ligase Chain Reaction (LCR)), high-density nucleic acid array-based processes, single nucleotide polymorphism (SNP) analyses, and nucleic acid sequencing techniques. To further illustrate, a number of methods for assaying other types of biomolecules that can utilize energy transfer to effect detection are also known. For example, proteins can be detected and quantified using various techniques, including SDS-polyacrylamide gel electrophoresis, capillary electrophoresis, enzyme assays, cell-based assays, and a wide range of immunological techniques, such as Western blotting and ELISA.
In addition, several diagnostic and analytical assays have been developed which involve the detection of multiple components in a sample using fluorescent dyes, including, e.g., flow cytometry (Lanier et al. (1984) “Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression,” J. Immunol. 132:151-156, which is incorporated by reference) and chromosome analysis (Gray et al. (1979) “High resolution chromosome analysis: one and two parameter flow cytometry,” Chromosoma 73:9-27, which is incorporated by reference), along with many of the assays referred to above. For these assays, it is desirable to simultaneously employ a set of two or more spectrally resolvable fluorescent dyes so that more than one target substance can be detected in the sample at the same time. Simultaneous detection of multiple components in a sample using multiple dyes reduces the time required to serially detect individual components in a sample. In the case of multi-loci DNA probe assays, the use of multiple spectrally resolvable fluorescent dyes reduces the number of reaction tubes that are needed, thereby simplifying the experimental protocols and facilitating the manufacturing of application-specific kits. In the case of automated DNA sequencing, for example, the use of multiple spectrally resolvable fluorescent dyes allows for the analysis of all four bases in a single lane thereby increasing throughput over single-color methods and eliminating uncertainties associated with inter-lane electrophoretic mobility variations.
Multiplex PCR detection using 5′ nuclease probes, molecular beacons, FRET or hybridization probes, among other multiplex detection methods, typically includes the pooling of quenched or unquenched fluorescent probes, e.g., to improve assay throughput relative to protocols that utilize single probes in a given reaction. To illustrate, multiplex assays are commonly used to detect multiple genotype markers or pathogens in samples obtained from patients as part of diagnostic procedures. In these formats, the overall baseline or background fluorescence from the pooled probes increases additively as the number of probes increases in the reaction mixture. This baseline fluorescence also increases in essentially any assay system when the amount of any single probe is increased. Baseline fluorescence generally adversely affects the performance of a given assay by, for example, reducing the detection sensitivity and dynamic range of the assay. Accordingly, baseline fluorescence effectively limits the total number of fluorescent probes and/or the amount of a given probe that can be utilized at one time in a particular assay.